Comparative Proteomics Research On BMSC Of Degenerative Scoliosis

Background:As the population aging trends going on and the lifestyle changing of the elderly, Degenerative scoliosis (DS) has become more and more important in the degenerative lumbar disease causing spinal deformity, dysfunction and affecting quality of life of the elderly. Although osteoporosis, asymmetric degenerative disc disease, facet tropism and inheritance have been implicated as factors in the development of DS, none of these has been shown to be directly related. The pathogenesis of DS is still unknown. Most current researches on the DS are focused on the therapeusis, but the basic research on the etiology is few. Furthermore, there has no molecular biology study been reported. So it is of great importance to find out the true reason or pathogenesis of DS.Bone merrow-derived mesenchymal stem cells(BM-MSCs) are the stem/progenitor cells of the osteoblasts. They can differentiate into several components of the bone, and the diseases associated with osteogenesis could be caused by the abnormity of the MSCs. So we should investigate the bone disease on the level of MSCs.Proteomics is a newly emerging experiment technology, it can find the differences on the level of the whole proteins to investigate the rule or nature of several vital movements and physiology or pathology phenomenons. The proteomics researches on the MSCs are just starting and rare, particularly the comparative proteomics researches on the disease. The recently introduced differential in gel electrophoresis(DIGE) technology is believed to be a highly reliable and accurate tool for quantitative analysis of differentially expressed proteins.In our study, MSCs of patients of DS and the Control(lumbar spinal stenosis) were isolated, cultured and identified. Then, the Comparative Proteomics Research on MSCs was performed to find the differential proteins which was studied and discussed to provide effective biochemical evidence for the coming gene researches and help to undersand the pathogenesis of DS.Objectives:1. To isolate and culture the MSCs of the patients of DS and the Control, and to identify the MSCs. 2. To establish two dimensional difference in gel electrophoresis(2D-DIGE) of MSCs of DS and the control.3. To identify the differential proteins expressed in MSCs of patients using mass spectrometry techniques.4. To analyze the role of the identified proteins in the development of degenerative scoliosis through the present protein functional network of research results.Methods:1. Bone marrow was collected from 12 degenerative scoliosis patients and 12 age-and gender-matched patients with lumbar spinal stenosis. Then the MSCs was harvested after being differentiated, cultured and identified.2. The samples were labeled with different CyDye, followed by 2D-DIGE, map scanning, and then the maps of protein spots were compared using specific software DeCyder v5.02.3. The differentially expressed spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and IPI-HUMAN data base.Results:l.By gradient centrifugation and adhesion culture, MSCs could be isolated and culture-expanded from human bone marrow aspirate. These cells were uniformly negative for CD31?CD34?CD45 and positive for CD29?CD44?CD105. The osteogenic differentiated cells were positive for alkaline phosphatase(ALP) and the adipogenic differentiated cells displayed accumulation of lipid vacuoles as detected by oil red O.2.115 spots that were differently expressed in the MSC of DS patients were found and identified. There were 70 proteins significantly up-regulated and 45 spots significantly down-regulated in MSC of DS patients.3. The differentially expressed spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS).44 proteins were identified from samples of DS and Control.Conclusions:1. MSCs can be isolated and expand-cultured from adult human bone marrow aspirate and committedly differentiate into osteoblasts and adipocytes. MSCs primary identification can be accomplished by flow cytometry and induced differentiation.2. This is the first time to study comparative proteomics on MSC of DS. And the MSC 2D-DIGE of DS patients and lumbar spinal stenosis were successfully settled.3. There were 44 protein spots of significance between the MSC of patients with DS and lumbar spinal stenosis analyzed by mass spectrometry techniques. Though the result is preliminary, but this differences of the protein may be the original factors of DS. Further investigations are necessary to illustrate the functioning pathway, the specificity and the mechanism of how these proteins contribute to pathogenesis of DS. The information obtained with this proteomic analysis will be very useful in understanding the pathophysiology of DS and finding the pathogenic genes of DS.